Conditional corticotropin-releasing hormone overexpression in the mouse forebrain enhances rapid eye movement sleep.

Impaired sleep and enhanced stress hormone secretion are the hallmarks of stress-related disorders, including major depression. The central neuropeptide, corticotropin-releasing hormone (CRH), is a key hormone that regulates humoral and behavioral adaptation to stress. Its prolonged hypersecretion is believed to play a key role in the development and course of depressive symptoms, and is associated with sleep impairment. To investigate the specific effects of central CRH overexpression on sleep, we used conditional mouse mutants that overexpress CRH in the entire central nervous system (CRH-COE-Nes) or only in the forebrain, including limbic structures (CRH-COE-Cam). Compared with wild-type or control mice during baseline, both homozygous CRH-COE-Nes and -Cam mice showed constantly increased rapid eye movement (REM) sleep, whereas slightly suppressed non-REM sleep was detected only in CRH-COE-Nes mice during the light period. In response to 6-h sleep deprivation, elevated levels of REM sleep also became evident in heterozygous CRH-COE-Nes and -Cam mice during recovery, which was reversed by treatment with a CRH receptor type 1 (CRHR1) antagonist in heterozygous and homozygous CRH-COE-Nes mice. The peripheral stress hormone levels were not elevated at baseline, and even after sleep deprivation they were indistinguishable across genotypes. As the stress axis was not altered, sleep changes, in particular enhanced REM sleep, occurring in these models are most likely induced by the forebrain CRH through the activation of CRHR1. CRH hypersecretion in the forebrain seems to drive REM sleep, supporting the notion that enhanced REM sleep may serve as biomarker for clinical conditions associated with enhanced CRH secretion.

Corticotropin-releasing hormone receptor type 1-deficiency enhances hippocampal serotonergic neurotransmission: an in vivo microdialysis study in mutant mice.

Corticotropin-releasing hormone plays an important role in the coordination of various responses to stress. Previous research has implicated both corticotropin-releasing hormone and the serotonergic system as causative factors in the development and course of stress-related psychiatric disorders such as major depression. To delineate the role of the corticotropin-releasing hormone receptor type 1 (CRH-R1) in the interactions between corticotropin-releasing hormone and serotonergic neurotransmission, in vivo microdialysis was performed in CRH-R1-deficient mice under basal (home cage) and stress (forced swimming) conditions. Hippocampal dialysates were used to measure extracellular levels of serotonin and its metabolite 5-hydroxyindoleacetic acid, and free corticosterone levels to monitor the status of the hypothalamic-pituitary-adrenocortical axis. Moreover, behavioural activity was assessed by visual observation and a scoring paradigm. Both wild-type and heterozygous mutant mice showed a clear diurnal rhythm in free corticosterone. Free corticosterone concentrations were, however, lower in heterozygous mutant mice than in wild-type animals and undetectable in homozygous CRH-R1-deficient mice. Homozygous CRH-R1-deficient mice showed enhanced hippocampal levels of 5-hydroxyindoleacetic acid but not of serotonin during the light and the dark phase of the diurnal cycle, which may point to an enhanced synthesis of serotonin in the raphe-hippocampal system. Moreover, the mutation resulted in higher behavioural activity in the home cage during the light but not during the dark period. Forced swimming caused a rise in hippocampal serotonin followed by a further increase after the end of the stress paradigm in all genotypes. Homozygous and heterozygous mutant mice showed, however, a significantly amplified serotonin response to the forced swimming as compared to wild-type control animals. We conclude that CRH-R1-deficiency results in reduced hypothalamic-pituitary-adrenocortical axis activity, in enhanced synthesis of serotonin during basal conditions, and in an augmented response in extracellular levels of serotonin to stress. These data provide further evidence for the intricate relationship between corticotropin-releasing hormone and serotonin and the important role of the CRH-R1 herein.

Glucocorticoid receptor impairment alters CNS responses to a psychological stressor: an in vivo microdialysis study in transgenic mice.

To study the consequences of impaired functioning of the glucocorticoid receptor (GR) for behavioural, neuroendocrine and neurochemical responses to a psychological stressor, a transgenic mouse expressing antisense RNA against GR was used. Previous studies on these transgenic mice have shown that impairment of GR evolves in disturbed neuroendocrine regulation and certain behavioural responses to stress. Here we investigated putative disturbances on the level of brain neurotransmission in GR-impaired (GR-i) mice using an in vivo microdialysis method. Through a microdialysis probe in the hippocampus, serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA) and free corticosterone [as an index of hypothalamic-pituitary-adrenocortical (HPA) axis activity] were monitored. Moreover, specific behaviours (e.g. grooming, eating/drinking, sniffing, nest building and locomotion) displayed by the mice during collection of the dialysates were scored. Measurement of dialysate concentrations of corticosterone on days 1 and 3 after insertion of the microdialysis probe showed that the free levels of this glucocorticoid were significantly lower in GR-i mice toward the evening. On day 2 after insertion of the microdialysis probe, baseline values of dialysate corticosterone, 5-HT and 5-HIAA were assessed, after which mice were exposed to a rat placed into their home cage. The rat and mouse were separated by a Plexiglas wall. A positive correlation between baseline hippocampal extracellular levels of 5-HT and 5-HIAA and the time spent performing active behaviours was observed in both genotypes. The main active behaviour performed at the baseline was grooming behaviour. During the rat exposure period, control mice remained mostly sitting and/or lying with their eyes fixed on the rat. Moreover, they showed a profound rise in free corticosterone levels. In contrast, GR-i mice displayed significantly more activities along the separation wall and a trend toward more grooming behaviour, but no increase of free corticosterone. In both mouse lines, exposure to a rat increased hippocampal extracellular levels of 5-HT and 5-HIAA. The rise in 5-HT was, however, more pronounced in the GR-i mice. From these data it may be concluded that life-long GR impairment has profound consequences for behavioural and neuroendocrine responses to a psychological stressor. Moreover, long-term impaired functioning of GR evolves in hyper-responsiveness of the raphe-hippocampal serotonergic system.

Long-term intracerebroventricular infusion of corticotropin-releasing hormone alters neuroendocrine, neurochemical, autonomic, behavioral, and cytokine responses to a systemic inflammatory challenge.

Corticotropin-releasing hormone (CRH) was infused intracerebroventricularly into rats for 7 d via a miniosmotic pump (1 microg . microl-1 . hr-1). Body temperature and locomotor activity were recorded during the treatment using biotelemetry, whereas hippocampal serotonergic neurotransmission and free corticosterone levels were monitored using in vivo microdialysis on day 7 of CRH treatment. During the microdialysis experiment, behavioral activity was scored by assessing the time during which rats were active (locomotion, grooming, eating, drinking). Continuous intracerebroventricular infusion of CRH produced a transient increase in body temperature and locomotion. Moreover, intracerebroventricularly CRH-treated rats showed elevated free corticosterone levels with no apparent diurnal rhythm. Intraperitoneal administration of bacterial endotoxin -lipopolysaccharide (LPS); 100 microg/kg body weight- on day 7 of CRH/vehicle treatment produced a marked fever response in control animals, which was significantly blunted in intracerebroventricularly CRH-treated rats. Although free corticosterone levels reached similar peak concentrations in both intracerebroventricularly vehicle- and CRH-infused groups after LPS, this response was delayed significantly by approximately 1 hr in the intracerebroventricularly CRH-treated animals. Microdialysis experiments showed no changes in basal extracellular levels of serotonin and 5-hydroxyindoleacetic acid in intracerebroventricularly CRH-infused animals. Injection of LPS in intracerebroventricularly CRH-treated rats produced a blunted 5-HT response and a delayed onset of behavioral inhibition and other signs of sickness behavior. Assessment of the endotoxin-induced cytokine responses showed significantly enhanced plasma interleukin-1 (IL-1) and IL-6 bioactivities in the intracerebroventricularly CRH-infused animals 3 hr after injection of LPS, whereas tumor necrosis factor bioactivity responses were not different. Our data demonstrate that chronically elevated brain CRH levels produce marked changes in basal (largely CRH regulated) physiological and behavioral processes accompanied by aberrant responses to an acute challenge. The present study provides evidence that chronic CRH hypersecretion is an important factor in the etiology of stress-related disorders.

Activation of serotonergic and noradrenergic neurotransmission in the rat hippocampus after peripheral administration of bacterial endotoxin: involvement of the cyclo-oxygenase pathway.

An endotoxic challenge produces pronounced effects on the immune, endocrine and central nervous systems. However, information on the brain structures and neurotransmitter systems participating in the physiological responses after stimulation of the immune system is still scarce. Using an in vivo microdialysis method is conscious, freely moving rats, the present study describes the effects of an endotoxic challenge on hippocampal serotonergic and noradrenergic neurotransmission. Rats were equipped with a microdialysis probe in the hippocampus, which enables the stress-free measurement of extracellular concentrations of serotonin, noradrenaline and their respective metabolites 5-hydroxyindoleacetic acid and 3-methoxy-4-hydroxyphenylglycol. The behavioral activity was scored by measurement of the time during which rats were active (locomotion, grooming, eating, drinking). In the control rats a significant, positive relationship between the behavioral activity and hippocampal extracellular levels of serotonin, noradrenaline and 3-methoxy-4-hydroxyphenylglycol was found. Intraperitoneally injected bacterial endotoxin (lipopolysaccharide; 100 micrograms/kg body weight) increased extracellular concentrations of serotonin, 5-hydroxyindoleacetic acid, noradrenaline and 3-methoxy-4-hydroxyphenylglycol, whereas the behavioral activity was largely reduced, thus disrupting the correlation between behavioral activity and hippocampal levels of serotonin, noradrenaline and 3-methoxy-4-hydroxyphenylglycol. Intraperitoneal pretreatment of rats with the cyclo-oxygenase inhibitor indomethacin attenuated, but did not completely abolish, the endotoxin-induced increases in hippocampal extracellular levels of serotonin, noradrenaline and their metabolites. From these results it may be concluded that the hippocampal serotonin and noradrenaline neurotransmitter systems are part of the brain circuitry responsive to an endotoxic challenge. Moreover, arachidonic acid metabolites seem to represent important, but not the sole, mediators of the endotoxin-induced changes in hippocampal neurotransmission.

Intraperitoneal administration of bacterial endotoxin enhances noradrenergic neurotransmission in the rat preoptic area: relationship with body temperature and hypothalamic--pituitary--adrenocortical axis activity.

A combined in vivo microdialysis/biotelemetry method in freely moving rats was used to study the effects of an endotoxic challenge on brain neurotransmission, hypothalamic-pituitary-adrenocortical (HPA) axis activity, autonomic functions and behaviour. Rats were equipped with a microdialysis probe in the preoptic area and a transmitter for biotelemetry in the peritoneal cavity. Time-dependent changes in noradrenergic and serotonergic neurotransmission, and HPA axis activity were monitored by measuring noradrenaline, serotonin, their metabolites and free corticosterone concentrations in dialysates. Core body temperature, heart rate and locomotion were measured simultaneously by biotelemetry. In addition, total behavioural activity was scored by measuring the time during which rats were active. Intraperitoneal administration of endotoxin (lipopolysaccharide; 100 micrograms/kg body weight) caused a pronounced increase in preoptic extracellular concentrations of noradrenaline and its metabolite 3-methoxy-4-hydroxyphenylglycol (MHPG; 500 and 400% of baseline respectively). No effect was found on preoptic concentrations of serotonin, although the levels of its metabolite 5-hydroxyindoleacetic acid were slightly elevated (120% of baseline). Intraperitoneal lipopolysaccharide caused a marked increase in corticosterone levels, a decline in behavioural activity, and biphasic rises in body temperature and heart rate. Analysis of the time curves revealed that noradrenaline rose in parallel with the first increase in body temperature and the increase in corticosterone levels. Moreover, maximum noradrenaline levels were reached approximately 60 min earlier than the peak in body temperature and corticosterone concentrations. Intraperitoneal pretreatment with the cyclo-oxygenase inhibitor indomethacin prevented the lipopolysaccharide-induced changes in body temperature, heart rate and behavioural activity, whereas the changes in noradrenaline, MHPG and corticosterone were largely, but not completely, reduced. Taken together, the results show that an endotoxic challenge results in a highly differentiated response in brain neurotransmission. We postulate that the profound increase in preoptic noradrenergic neurotransmission may be related to the lipopolysaccharide-evoked induction of fever and/or activation of the HPA axis.

Effect of bacterial endotoxin and interleukin-1 beta on hippocampal serotonergic neurotransmission, behavioral activity, and free corticosterone levels: an in vivo microdialysis study.

In this study the effect of immune system stimulation and intracerebroventricular (i.c.v.) administration of interleukin-1 beta (IL-1 beta) on hippocampal serotonergic neurotransmission, behavioral activity, and the hypothalamic-pituitary-adrenocortical (HPA) axis is described. An in vivo microdialysis method was used to measure hippocampal extracellular concentrations of serotonin (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) in conscious, freely moving rats. In addition, we established a method to continuously monitor free corticosterone levels in dialysates. Behavioral activity was scored by measuring the time during which rats were active (locomotion, grooming, eating, drinking). We found a significant, positive relationship between behavioral activity and hippocampal extracellular concentrations of 5-HT. Intraperitoneal (i.p.) administration of the bacterial endotoxin lipopolysaccharide (LPS; 30, 100, and 300 micrograms/kg body weight) produced an increase in the extracellular concentrations of 5-HT and 5-HIAA in the hippocampus, which was paralleled by a significant decline in behavioral activity and a marked increase in extracellular corticosterone levels. Thus, the close correlation between hippocampal extracellular 5-HT levels and behavioral activity observed in control rats was disrupted in the LPS-treated animals. The effects of i.p. LPS could be mimicked by i.c.v. application of recombinant human IL-1 beta (hIL-1 beta; 100 ng). i.c.v. pretreatment with the IL-1 receptor antagonist (IL-1ra; 10 micrograms) antagonized the hIL-1 beta-induced effects. IL-1ra showed no intrinsic effects. Furthermore, it was found that i.c.v. pretreatment with IL-1ra (10 micrograms) significantly attenuated the i.p. LPS-induced (100 micrograms/kg body weight) rise in hippocampal extracellular 5-HT levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Neural encoding of amplitude modulation within the auditory midbrain of squirrel monkeys.

The neuronal responses to amplitude modulated (AM) sounds were investigated in the auditory midbrain of the squirrel monkey. Sinusoidally modulated tones and noise served as acoustic stimuli. In order to describe the response properties of collicular neurons, Fast-Fourier-Transformation (FFT), a cross-correlation algorithm and spike-rate counts were applied to translate the neuronal reactions into modulation transfer functions. FFT and cross-correlation defined a measure for synchronicity of the neuronal discharges with the modulation cycles. All neurons (542) responded selectively to AM-sounds insofar as all displayed a best modulation frequency (BMF). Most of them furthermore had a band-pass-like modulation transfer function, whose center frequencies were mainly between 8 and 128 Hz. Transfer functions obtained by spike-rate showed less selectivity: a relatively great number of neurons did not change their spike rate as a function of modulation frequency. The results show that encoding of amplitude-modulated sounds occurs to a greater extent via phase locking of discharges than via changes in spike number. In the same way, changing modulation depth is processed: whereas spike rate on average remains constant between 100% and 0% modulation, there is a drastic reduction in synchronicity. No clear relationship was found between a unit's characteristic frequency and BMF; the same applied to BMF and recording place. The results furthermore show that amplitude modulations are encoded selectively in a band pass function in a non-human primate. The midbrain thereby occupies an intermediate position within the pathway from the periphery to the cortex. This form of temporal resolution probably underlies mechanisms caused by the increasing synaptic activity in the course of the pathway. This may indicate adaptation since those modulation frequencies embedded in this species' vocal repertoire fit quite well with the system's tuning properties for amplitude modulation.

Local administration of recombinant human interleukin-1 beta in the rat hippocampus increases serotonergic neurotransmission, hypothalamic-pituitary-adrenocortical axis activity, and body temperature.

In this study, we equipped rats with a microdialysis probe in the hippocampus, which enabled stress-free intrahippocampal administration of recombinant human IL-1 beta (hIL-1 beta). Perfusion of the probes was conducted with a Ringer's solution containing 0.1 or 1.0 microM hIL-1 beta or without hIL-1 beta, usually for 6 h. Time-dependent changes in serotonergic neurotransmission and hypothalamic-pituitary-adrenocortical activity were simultaneously monitored by measuring serotonin [5-hydroxytryptamine (5-HT)], 5-hydroxyindoleacetic acid, and corticosterone concentrations in the dialysates. In control rats, there was a clear relationship between extracellular 5-HT concentrations in the hippocampus and behavioral activity. Extracellular 5-HT levels were up to twice as high in behaviorally active rats compared to those in resting or sleeping animals. Intrahippocampal administration of hIL-1 beta markedly increased extracellular 5-HT concentrations in the hippocampus and induced a significant decrease in behavioral activity, thereby uncoupling the parallelism between changes in 5-HT and changes in behavioral activity observed in control rats. Perfusion with 0.1 microM hIL-1 beta, but not with 1 microM hIL-1 beta, produced a decrease in 5-hydroxyindoleacetic acid levels, followed by a return to preinfusion levels. Moreover, intrahippocampal administration of hIL-1 beta increased hypothalamic-pituitary-adrenocortical axis activity, as evidenced by marked increases in both plasma ACTH and plasma and dialysate corticosterone levels. In addition, a rise in body temperature by approximately 2 C was observed at time points at which the effects of hIL-1 beta on 5-HT and corticosterone levels were (near-)maximal. hIL-1 beta-treated rats displayed typical characteristics of sickness behavior, such as immobility, piloerection, and a curled-up body posture. Most importantly, no effects were found either with heat-inactivated hIL-1 beta or when hIL-1 beta was administered via a probe implanted in the neocortex. Based on these results, we postulate that the hippocampal IL-1 system may play an important role in the coordination of neuroendocrine, autonomic, and behavioral responses after an immune challenge.